Red blood cells (RBC) are the oxygen delivery component of the human body and play a major role in maintaining and supporting several physiological systems that are critically important in sustaining life. Conditions that affect the health or sufficiency of the body's supply of RBC are always significant to a patient and may be life threatening.
Several characteristics of RBCs are critical. The RBCs must maintain the capability to deliver oxygen and their structural and mechanical integrity must not be compromised. Sickle cell anemia is a conspicuous example of a serious pathological condition characterized by abnormal rigidity of RBCs.
Furthermore, many diseases and medical procedures require supplementing a patient's blood supply with RBCs from a donor--typically a human source. The RBCs must be compatible with the patient. For example, the need to determine blood type and antigenicity cross-matching is a result of the requirement that RBCs must not be recognized as a foreign substance by the immune system of the patient. Where the patient's immune system attacks an infusion of RBCs, serious transfusion reactions result.
There are currently over 250 known blood group antigens, most of which are classified into 23 groups. Of these, the A, B, O, and Rh groups are of greatest significance for blood transfusion. Antibodies to A and B are naturally occurring in the majority of recipients. Rh D antigen is strongly immunogenic in Rh D negative recipients. Thus, it is universal practice to type the recipient for the A, B antigens and, except in unusual circumstances, the Rh D antigen prior to transfusion to ensure that compatible donor blood is given. The recipient's plasma is also screened to determine whether it contains alloantibodies to one or more of the minor blood group antigens on the donor RBC. The presence of an antibody which recognizes the donor cells results in the transfusion reaction mentioned above, in which the transfused RBC are rapidly destroyed. Alloantibodies develop because it is impossible to obtain a perfect match between recipient and donor with each possible combination of blood group antigens, and are most likely to develop in patients who have received multiple previous transfusions.
Still further interruptions or changes in flow characteristics of RBCs can exert profound effects on the body. Heart attacks, strokes, and other conditions accompanied by ischemia and/or reperfusion injury are among the most serious medical conditions affecting the population. The characteristics of blood flow are determined to a great degree by the viscosity of blood as it flows throughout the cardiovascular system. Viscosity is a measure of the resistance to flow and the viscosity of human blood dependent on several factors, including interactions between RBCs in blood. The higher the viscosity, the greater the force that is required to maintain a given blood flow rate.
The viscosity of human blood is highly shear-rate dependent (non-Newtonian). At high shear rates, the viscosity tends toward a minimum value, which depends upon the plasma viscosity, the volume fraction of red blood cells (hematocrit) and the deformability of each individual RBC. At high shear rates, there are no significant interactions between the RBCs. At low shear rates, the viscosity increases substantially because the RBCs aggregate together, effectively forming larger particles. This aggregation phenomenon is mediated by plasma proteins, primarily fibrinogen, but also immunoglobulins, and is completely reversible upon increasing the shear rate. The shear-rate dependence of blood viscosity leads to a disproportionate decrease in blood flow at low flow rates, such as may occur during hemorrhagic shock, or after ischemic injury to tissues. The physiological significance is that the decrease in blood flow further reduces the delivery of oxygen and nutrients to the tissues, and potentially worsens the ischemic injury.
Hemodilution is the preferred therapeutic option for reduction of blood viscosity. Hemodilution is achieved by intravenous infusion of human albumin, or a hydrocolloid polymer solution, e.g., dextran or a modified starch, such as HES (hydroxyethyl starch), as a plasma volume expander. Hemodilution has been shown to be effective in the management of acute or chronic ischemia, such as stroke, cerebral vasospasm, critical limb ischemia and peripheral vascular disease. Blood viscosity is reduced due a combination of the reduced hematocrit and dilutional effect on the plasma fibrinogen concentration. In general, the practice of hemodilution in clinical applications aims to lower the hematocrit to between 0.30 and 0.35, at which level the reduction in O.sub.2 carrying capacity by the RBCs is offset by increased blood flow due to flow resistance and a consequent increase in cardiac output. However, hemodilution is not an appropriate option for disorders such as myocardial ischemia in which cardiac reserve may already be compromised, pre-existing cardiac failure, or sickle cell disease (pre-existing anemia). In such cases, an alternative means to reduce blood viscosity without significant hemodilution would be desirable.
Polyethylene glycol (PEG) is an amphiphilic linear polymer, which is non-mimmunogenic, non-toxic and chemically inert in biological systems. PEG-modified biomolecules such as albumin have been shown to be intrinsically less immunogenic and to have a prolonged circulation time in rats whose immune systems have been pre-sensitized with unmodified bovine albumin. The covalent attachment of PEG is now commonly used to modify many proteins, enzymes, drugs and artificial surfaces that come into contact with human blood. PEG-modified enzymes are in clinical use, e.g., PEG-adenosine deaminase for the treatment of severe combined immunodeficiency, and PEG-modified hemoglobins have been developed for use as hemoglobin-based oxygen carriers (HBOC) as blood substitutes. Liposomes coated with PEG have been evaluated for use both as artificial hemoglobin carriers and as drug delivery systems.
Despite these seemingly diverse applications, essentially two reasons exist for modifying a protein or biomolecule with PEG. First, a potentially antigenic protein or enzyme labelled with PEG is not susceptible to attack by the immune system. The extremely hydrophilic nature of the PEG molecule, which in aqueous solution is surrounded by a large volume of coordinated water molecules to establish a hydrodynamic radius, contributes to the reduced antigenicity of PEG-labelled biomolecules. Once the PEG molecule is attached to a biomolecule, such as a protein near to a potentially antigenic site, the PEG molecule with its associated hydration sphere sterically hinders the approach of antibodies or other immunoproteins. Second, PEG-modification alters the physical properties of the substance to which the PEG is attached. For example, in PEG-modified bovine hemoglobin, aside from reducing the potential antigenicity of the bovine protein, the PEG increases the molecular weight of the hemoglobin, which reduces extravasation and slows the clearance from the blood stream and reduces renal toxicity. In the case of PEG-modified liposomes, the PEG-coating increases the hydrophilicity of the surface, prevents aggregation (flocculation) of the particles in suspension, and significantly delays uptake by the reticuloendothelial system. PEG molecules are also covalently bound to plastic surfaces to improve biocompatibility and to solubilize pharmaceutical agents that would otherwise be too hydrophobic for use.
Therefore, the use of reactive PEG intermediates has recently been widely applied to modify synthetic surfaces, proteins, liposomes, and drugs. The PEG coating of these substances has enabled prolonged circulatory times, increased biocompatibility, and reduced immunogenicity. However, because most PEG-modification techniques require highly non-physiological conditions, the direct bonding of PEG to living cells has not been practical nor widely practiced.
Poloxamer 188 (P188), also known as Pluronic F68, is an agent that inhibits RBC aggregation and reduces blood viscosity in vitro. A pharmaceutical preparation of P188 (RheothRx.RTM. injection) has been shown to improve blood flow in ischemic tissues and to reduce myocardial infarct size in animal models. Recent clinical studies have demonstrated significant potential for RheothRx.RTM. in the treatment of myocardial infarction and sickle cell crisis.
The poloxamer molecule consists of two hydrophilic poly(ethylene glycol) (PEG) chains connected by a hydrophobic poly(propylene glycol) (PPG) core to form an A-B-A triblock copolymer of PEG(A) and PPG(B) having a total molecular weight of approximately 8400 Daltons (80% PEG and 20% PPG). The mechanism of action of P188 appears to result from adsorption of the hydrophobic PPG core onto the RBC surface, with the hydrophilic PEG segments extending outward from the cell surface, forming a steric barrier which inhibits RBC aggregation and consequently reduces low shear blood viscosity by preventing cell-cell or cell-plasma interactions. However, P188 suffers from certain drawbacks. A relatively high plasma concentration of P188 (&gt;1 mg/ml) is needed to achieve a significant reduction of RBC aggregation. As P188 undergoes rapid renal clearance from the circulation (t.sub.1/2 =5 hr), a continuous intravenous infusion of 30-60 mg/kg/hr is required to maintain a therapeutic plasma level, which amounts to a total dose of 50 g/day or more. Therefore, P188 is somewhat inefficient (low potency) at lower concentrations and is toxic at concentrations high enough to yield significant benefits.
These disadvantages are a consequence of the very weak hydrophobic interaction between the hydrophobic PPG and the RBC, and the small size of the molecule, which causes it to be excreted very rapidly (half-life 2-5 hours). This disadvantage cannot be remedied by increasing the size or the relative proportions of PEG and PPG in the molecule--Pluronics larger than F68, e.g. F108 and F127, which have a larger hydrophobic segment and which might be expected to bind more strongly to RBC, tend to self-associate when added to blood, and thus promote, rather than inhibit, red cell aggregation.